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PubMed1993

Effect of mutations in SOS genes on UV-induced precise excision of Tn10 in Escherichia coli.

UV treatment increases the frequency of Tn10 precise excision from different sites of the Escherichia coli chromosome. UV induction of Tn10 excision is not evidenced in a lexA3 (ind-) mutant carrying either a recA+ or a recA730 allele. High levels of RecA synthesized by a recA+ gene not repressible by LexA do not relieve the non-inducibility of Tn10 excision in a lexA3 (ind-) background. This indicates that the expression of an SOS gene different from recA is necessary for the induction of Tn10 excision. In contrast to UV induction of point mutations, this induction does not depend on a functional umuC gene since umuC::Tn5 mutants show increased levels of Tn10 excision from leu, thr or gal after irradiation. MucAB+ plasmid pKM101 which renders cells more UV-mutable for point mutations decreases UV-induced Tn10 excision. These results show that UV-induced Tn10 precise excision requires SOS induction and that it involves a pathway different from point mutagenesis.

Bacterial Proteins↗
PubMed1997

Mycobacterial recA is cotranscribed with a potential regulatory gene called recX.

The recA gene of Mycobacterium smegmatis has been cloned and sequenced. The amino acid sequence of the RecA protein is highly homologous to other RecA proteins. Three other potential open reading frames were identified. One of these showed extensive homology to a protein, HypB, involved in the incorporation of nickel into hydrogenases. Another, found downstream of and overlapping recA, was similar to a gene, recX, which has been proposed to play a regulatory role related to recA function. The homology between the M. smegmatis sequence and that of Mycobacterium tuberculosis extended upstream of the recA coding region for 140 bp including a motif identical to the Cheo-box consensus sequence which has been shown to bind LexA. In addition, the transcriptional start sites were found to be identical to those identified previously for M. tuberculosis. Transcriptional fusions to the reporter gene chloramphenicol acetyltransferase (CAT) revealed that recA was DNA-damage inducible and that expression required sequences at some distance from the mapped transcriptional start sites. Although a motif with only one mismatch to the Cheo box was found in the intergenic region between orf1 and orf2 these open reading frames were not DNA-damage inducible, nor was this motif required for regulation of recA expression. Gel retardation assays revealed that the reason for this was that LexA did not bind to this sequence containing a mismatch. Reverse transcription/polymerase chain reaction analysis of M. smegmatis RNA demonstrated that recA and orf3 (recX) are within the same transcriptional unit.

Amino Acid Sequence↗
PubMed2001

Growth-dependent DNA breakage and cell death in a gyrase mutant of Salmonella.

A class of gyrase mutants of Salmonella enterica mimics the properties of bacteria exposed to quinolones. These mutants suffer spontaneous DNA breakage during normal growth and depend on recombinational repair for viability. Unlike quinolone-treated bacteria, however, they do not show accumulation of cleavable gyrase-DNA complexes. In recA or recB mutant backgrounds, the temperature-sensitive (ts) allele gyrA208 causes rapid cell death at 43 degrees. Here, we isolated "suppressor-of-death" mutations, that is, secondary changes that allow a gyrA208 recB double mutant to survive a prolonged exposure to 43 degrees and subsequently to form colonies at 28 degrees. In most isolates, the secondary change was itself a ts mutation. Three ts alleles were mapped in genes coding for amino acyl tRNA synthetases (alaS, glnS, and lysS). Allele alaS216 completely abolished DNA breakage in a gyrA208 recA double mutant. Likewise, treating this mutant with chloramphenicol prevented death and DNA damage at 43 degrees. Additional suppressors of gyrA208 lethality include rpoB mutations and, surprisingly, icd mutations inactivating isocitrate dehydrogenase. We postulate that the primary effect of the gyrase alteration is to hamper replication fork movement. Inhibiting DNA replication under conditions of continuing macromolecular synthesis ("unbalanced growth") activates a mechanism that causes DNA breakage and cell death, reminiscent of "thymineless" lethality.

Alleles↗
PubMed1999

Role of Escherichia coli RpoS, LexA and H-NS global regulators in metabolism and survival under aerobic, phosphate-starvation conditions.

It has been suggested that Escherichia coli can resist aerobic, glucose-starvation conditions by switching rapidly from an aerobic to a fermentative metabolism, thereby preventing the production by the respiratory chain of reactive oxygen species (ROS) that can damage cellular constituents. In contrast, it has been reported that E. coli cannot resist aerobic, phosphate (Pi)-starvation conditions, probably because of the maintenance of an aerobic metabolism and the continuous production of ROS. This paper presents evidence that E. coli cells starved for Pi under aerobic conditions indeed maintain an active aerobic metabolism for about 3 d, which allows the complete degradation of exogenous nutrients such as arginine (metabolized probably to putrescine via the SpeA-initiated pathway) and glucose (metabolized notably to acetate), but cell viability is not significantly affected because of the protection afforded against ROS through the expression of the RpoS and LexA regulons. The involvement of the LexA-controlled RuvAB and RecA proteins with the RecG and RecBCD proteins in metabolism and cell viability implies that DNA double-strand breaks (DSB), and thus hydroxyl radicals that normally generate this type of damage, are produced in Pi-starved cells. It is shown that induction of the LexA regulon, which helps protect Pi-starved cells, is totally prevented by introduction of a recB mutation, which indicates that DSB are actually the main DNA lesion generated in Pi-starved cells. The requirement of RpoS for survival of cells starved for Pi may thus be explained by the role played by various RpoS-controlled gene products such as KatE, KatG and Dps in the protection of DNA against ROS. In the same light, the degradation of arginine and threonine may be accounted for by the synthesis of polyamines (putrescine and spermidine) that protect nucleic acids from ROS. Besides LexA and RpoS, a third global regulator, the nucleoid-associated protein H-NS, is also shown to play a key role in Pi-starved cells. Through a modulation of the metabolism during Pi starvation, H-NS may perform two complementary tasks: it helps maintain a rapid metabolism of glucose and arginine, probably by favouring the activity of aerobic enzymes such as the NAD-dependent pyruvate dehydrogenase complex, and it may enhance the cellular defences against ROS which are then produced by increasing RpoS activity via the synthesis of acetate and presumably homoserine lactone.

Aerobiosis↗
PubMed1985

Identification of the Escherichia coli recN gene product as a major SOS protein.

The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified.

Bacterial Proteins↗
PubMed1993

Characterization of dinY, a new Escherichia coli DNA repair gene whose products are damage inducible even in a lexA(Def) background.

Bacteriophage Mu dX(Ap lac) was used to isolate a mutation in an Escherichia coli lexA(Def) strain representing a previously undescribed gene (dinY) which does not seem to be under the direct control of LexA. The insertion created a dinY::lacZ fusion in which beta-galactosidase expression required a DNA-damaging treatment (UV irradiation or mitomycin) and activable RecA protein. This strain showed a decreased Weigle reactivation of bacteriophage lambda. However, it was fully inducible for UV mutagenesis. Two-dimensional gel electrophoresis analysis identified two spots absent in the mutant which were both UV inducible only in the presence of activated RecA protein (RecA*). This finding suggests that the dinY::lacZ fusion lies in a gene either that is under the direct control of activated RecA or whose product undergoes RecA*-dependent posttranscriptional/posttranslational modification(s). The dinY gene may also control the expression of some other gene(s) and/or lie in an operon. The fusion was mapped at a position between 41 and 41.5 min on the E. coli chromosome, in the vicinity of the ruv operon.

Bacterial Proteins↗
PubMed1996

Multicopy suppressors of the cold-sensitive phenotype of the pcsA68 (dinD68) mutation in Escherichia coli.

The Escherichia coli strain cs2-68 is a cold-sensitive (c) mutant that forms a long filamentous cell at 20 degrees C with a large nucleoid mass in its central region. We have recently shown that the pcsA68 mutation causing the cs phenotype is a single-base substitution within the dinD gene, a DNA damage-inducible gene which maps at 82 min. Since null mutants of the pcsA (dinD) gene are viable, with no discernible defect in cell growth, the cs phenotype is attributed to a toxic effect by the mutant protein. In an attempt to identify a target(s) for the toxic pcsA68 mutant protein, we screened for chromosomal fragments on multicopy plasmids that could suppress the cs phenotype. Three different BamHI fragments were found to suppress cold sensitivity, and the lexA, dinG, and dinI genes were identified to be responsible for the suppression in each fragment. DinG shares multiple motifs with many DNA helicases. The complete sequence of dinI revealed that DinI is a small protein of 81 amino acids. It is similar in size and sequence to ImpC of the Salmonella typhimurium plasmid TP110 and to a protein (ORFfs) of the retronphage phi R67, both of which are also under the control of LexA.

Amino Acid Sequence↗
PubMed2003

Characterization of a new LexA binding motif in the marine magnetotactic bacterium strain MC-1.

MC-1 is a marine, magnetotactic bacterium that is phylogenetically associated with the alpha subclass of the Proteobacteria and is the first and only magnetotactic coccus isolated in pure culture to date. By using a TBLASTN search, a lexA gene was identified in the published genome of MC-1; it was subsequently cloned, and the protein was purified to >90% purity. Results from reverse transcription-PCR analysis revealed that the MC-1 lexA gene comprises a single transcriptional unit with two open reading frames encoding proteins of unknown function and with a rumA-like gene, a homologue of the Escherichia coli umuD gene. Mobility shift assays revealed that this LexA protein specifically binds both to its own promoter and to that of the umuDC operon. However, MC-1 LexA does not bind to the promoter regions of other genes, such as recA and uvrA, that have been previously reported to be regulated by LexA in bacterial species belonging to the alpha subclass of the Proteobacteria: Site-directed mutagenesis of both the lexA and umuDC operator regions demonstrated that the sequence CCTN(10)AGG is the specific target motif for the MC-1 LexA protein.

Alphaproteobacteria↗
PubMed1994

Identification of residues in the L1 region of the RecA protein which are important to recombination or coprotease activities.

Using a combinatorial cassette mutagenesis procedure we have introduced a large number of single and multiple amino acid substitutions into an area of the RecA protein defined by residues 152-159. This sequence overlaps the disordered loop 1 region (L1) in the RecA crystal structure which has been hypothesized to be involved in DNA binding. Assays for recombinational DNA repair and LexA coprotease activities identify Glu154 as the only one of these 8 residues which is critical to RecA function. Several other mutations observed at nearby residues support the identity of Glu154 as the most important of the 14 residues in the area defined by Pro151 to Met164. In addition, Gly157 and Glu158 appear to be hot spots for the occurrence of mutation-induced constitutive coprotease activity.

Amino Acid Sequence↗
PubMed1996

The proto-oncogene HLF and the related basic leucine zipper protein TEF display highly similar DNA-binding and transcriptional regulatory properties.

Genes encoding transcription factors are frequently altered by chromosomal translocations in acute lymphoblastic leukemia (ALL), suggesting that aberrant transcriptional regulation plays a prominent role in leukemogenesis. E2A-hepatic leukemia factor (HLF), a chimeric transcription factor created by the t(17;19), consists of the amino terminal portion of E2A proteins, including two experimentally defined transcriptional activation domains (TADs), fused to the HLF DNA binding and protein dimerization basic leucine zipper (bZIP) domain. To understand the mechanisms by which E2A-HLF induces leukemia and the crucial functions contributed by each constituent of the chimera, it is essential to define the normal transcriptional regulatory properties of HLF and related bZIP proteins. To address these questions, we cloned the human homologue of TEF/VBP, a bZIP protein closely related to HLF. Using a binding site selection assay, we found that TEF bound preferentially to the consensus sequence 5'-GTTACGTAAT-3', which is identical to the previously determined HLF recognition site. TEF and HLF activated transcription of consensus site-containing reporter genes in several different cell types with similar potencies. Using GAL4 chimeric proteins, a TAD was mapped to a discrete approximate 40 amino acid region of TEF and HLF within which they share 72% amino acid identity and 85% similarity. The TEF/HLF activation domain (THAD) has a predicted helical secondary structure, but shares no sequence homology with previously reported TADs. The THAD contained most, if not all, of the transcriptional activation properties present in both TEF and HLF and its deletion completely abrogated transcriptional activity of TEF and HLF in both mammalian cells and yeast. Thus, TEF and HLF share indistinguishable DNA-binding and transcriptional regulatory properties, whose alteration in leukemia may be pathogenetically important.

Amino Acid Sequence↗
PubMed1985

Mechanism of transient inhibition of DNA synthesis in ultraviolet-irradiated E. coli: inhibition is independent of recA whilst recovery requires RecA protein itself and an additional, inducible SOS function.

The mechanism of the inhibition and of the recovery of DNA synthesis in E. coli following UV-irradiation was analysed in several mutants defective in repair or in the regulation of the RecA-LexA dependent SOS response. Several lines of evidence indicated that inhibition is not an inducible function and is probably due to the direct effect of lesions in the template blocking replisome movement. Recovery of DNA synthesis after UV was largely unaffected by mutations in the uvrA, recB or umuC genes. Resumption of DNA synthesis does however require protein synthesis and the regulatory action of recA. Experiments with a recA constitutive mutant and recA 200 (temperature sensitive RecA) demonstrated that RecA protein itself is directly required but is not sufficient for recovery of DNA synthesis. We therefore propose that recovery of DNA synthesis depends upon the concerted activity of RecA and the synthesis of an inducible Irr (induced replisome reactivation) factor under RecA control. We suggest that the mechanism of recovery involves the action of Irr and RecA to promote movement of replisomes past non-instructive lesions, uncoupled from polymerisation and/or that Irr and RecA are required to promote re-initiation of a stalled replication complex downstream of a UV-lesion subsequent to such an uncoupling step.

Bacterial Proteins↗
PubMed1985

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli III. Enhancement of excision repair in UV-treated bacteria.

The question of whether induction of the SOS response in Escherichia coli increases the efficiency of excision repair was addressed by measuring repair of UV-damaged nonreplicating lambda phage DNA in previously irradiated bacteria. Prior UV irradiation of lex+ bacteria enhanced both the rate of regeneration of infective phage DNA (about 10-fold) and the rate of cyclobutane dimer removal early in repressed infections. Indirect induction of SOS-regulated repair activities by the nonreplicating irradiated phage DNA itself seemed negligible. Prior bacterial irradiation reduced the frequency of recombination (loss of a tandem chromosomal duplication) of nonreplicating UV-irradiated DNA. In this respect UV-stimulated recombination of nonreplicating DNA differs from RecF-dependent recombination processes that are stimulated by increased SOS expression. Surprisingly, prior UV irradiation of lexA3 bacteria caused a small but reproducible increase in the regeneration of infective phage DNA.

Bacterial Proteins↗
PubMed1996

Two overlapping SOS-boxes in ColE operons are responsible for the viability of cells harboring the Col plasmid.

In this study, oligonucleotide-directed site-specific mutagenesis was used to change the consensus sequences of the LexA binding motifs in either one of the two SOS-boxes of the ColE7 operon. The results indicated that both mutants produced larger amounts of colicin than cells harboring the wild-type ColE7 plasmid. This finding would imply that two biologically functional SOS boxes exist in the ColE7 operon. In the non-induced state, no lysis of cells harboring wild-type plasmids occurred at 37 degrees C, whereas, cells harboring recombinant plasmids containing either one of the mutated SOS boxes underwent lysis within 100 min under the same conditions. This result indicated that adaptation of two SOS boxes of the ColE operon would obviously tightly control the expression of ColE operons. In such a way that it may prevent excessive expression of the lysis (cel) gene, thus safeguard the host cells from being lysed in ordinary living conditions.

Bacterial Proteins↗
PubMed1985

Interspecies regulation of the SOS response by the E. coli lexA+ gene.

A plasmid-encoded E. coli lexA+ gene was introduced into 6 species of Enterobacteria. Ultraviolet light-sensitization occurred in all species except P. rettgeri, and 4 organisms showed reduced inducibility of RecA-like proteins. The mechanism of lexA+ control of the SOS response therefore appears common to several species.

Bacterial Proteins↗
PubMed1980

Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12.

The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322. Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA. The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment. Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated. To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4. A restriction map of pLC44-14 was obtained for nine restriction enzymes. The orientation of this map was determined relative to the E. coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.

Bacterial Proteins↗
PubMed1997

Enhanced generation of A:T-->T:A transversions in a recA730 lexA51(Def) mutant of Escherichia coli.

RecA730 belongs to a class of mutant RecA protein that is often referred to as RecA*, since it is constitutively activated for coprotease functions in the absence of exogenous DNA-damage. Escherichia coli strains carrying recA730 (or other recA* alleles) exhibit dramatic increases in SOS-dependent spontaneous mutator activity. We have analyzed the specificity of this mutator phenotype by employing F'-plasmids carrying a set of mutant lacZ genes that can individually detect two types of transitions, four types of transversions, and four kinds of specific frameshift events. Analysis revealed that most of the spontaneous mutagenesis in a recA730 lexA51(Def) strain (which expresses derepressed levels of all LexA-regulated proteins) can be attributed to a specific increase in A:T-->T:A, A:T-->C:G and G:C-->T:A transversions, with the A:T-->T:A transversions occurring most frequently. These transversion events were completely abolished in a delta umuDC strain, indicating that the functionally active UmuD'C proteins are normally required for their generation. The spectrum obtained was similar to that of strains with a defect in the epsilon (3'-->5' proofreading) subunit of DNA polymerase III. Such an observation raises the possibility that the wild-type epsilon protein is in activated in strains expressing the RecA730 and UmuD'C proteins.

Bacterial Proteins↗
PubMed1990

Identification of consensus patterns in unaligned DNA sequences known to be functionally related.

We have developed a method for identifying consensus patterns in a set of unaligned DNA sequences known to bind a common protein or to have some other common biochemical function. The method is based on a matrix representation of binding site patterns. Each row of the matrix represents one of the four possible bases, each column represents one of the positions of the binding site and each element is determined by the frequency the indicated base occurs at the indicated position. The goal of the method is to find the most significant matrix--i.e. the one with the lowest probability of occurring by chance--out of all the matrices that can be formed from the set of related sequences. The reliability of the method improves with the number of sequences, while the time required increases only linearly with the number of sequences. To test this method, we analysed 11 DNA sequences containing promoters regulated by the Escherichia coli LexA protein. The matrices we found were consistent with the known consensus sequence, and could distinguish the generally accepted LexA binding sites from other DNA sequences.

Algorithms↗